Quinolones, such as ciprofloxacin and levofloxacin (Levaquin(R)), are the most efficacious and broad- spectrum oral antibacterials currently in clinical use. These drugs are used as front-line treatments for a wide variety of human infections caused by Gram-negative and Gram-positive bacteria. The cellular targets of quinolones are the bacterial type II topoisomerases, gyrase and topoisomerase IV. These closely related enzymes are essential to cell survival. Gyrase regulates levels of DNA under- and over- winding and alleviates torsional stress that accumulates ahead of DNA replication forks and transcription complexes. Topoisomerase IV alleviates torsional stress, removes knots and tangles from the genome, and is required for proper chromosomal partitioning. Both enzymes function by passing an intact double helix through a transient double-stranded break that they generate in a separate DNA segment. Quinolones kill bacteria by increasing levels of DNA strand breaks generated by gyrase and topoisomerase IV, effectively converting these enzymes into potent cellular toxins that fragment the genome. Both type II enzymes are physiological targets for quinolones, but their relative importance to drug action appears to be species- and drug-dependent. There is a growing crisis in antibacterial resistance, and quinolone resistance is becoming prevalent. Initial quinolone resistance is most often associated with specific mutations in gyrase and/or topoisomerase IV. Resistance can range from <10- to >100-fold, depending on whether one or both enzymes are mutated. The most common resistance mutations occur at a serine residue originally described as Ser83 in the GyrA subunit of Escherichia coli gyrase and a glutamic/aspartic acid residue 4 amino acids downstream. Based on functional studies from the Osheroff laboratory and a published structure, these residues are proposed to anchor a water- metal ion bridge that serves as the primary conduit between quinolones and the type II enzymes. Despite the wide clinical use of quinolones, relatively little is understood regarding their interactions with gyrase or topoisomerase IV. Although amino acid mutations in gyrase and topoisomerase IV that are associated with drug resistance have been documented in thousands of isolates, only a handful of studies have gone beyond identifying mutated residues and addressed the underlying biochemical basis for quinolone resistance. Clearly, there is an urgent need to develop more effective drugs that display activity against resistant strains. Therefore, to address this critical issue, the specific aims of this proposal are: 1) To determine the mechanistic basis for quinolone action and resistance. A variety of biochemical and biophysical approaches will be used. Studies will focus on elucidating quinolone-enzyme interactions and the role of the proposed water-metal ion bridge. 2) To determine the roles of gyrase and topoisomerase IV in mediating quinolone-induced toxicity. Relationships between quinolone potency, efficacy, and resistance in vitro and the relative importance of gyrase vs. topoisomerase IV in mediating drug-induced cell kill will be defined. We will determine physiological levels of quinolone-induced DNA cleavage mediated by both enzymes and the persistence of gyrase- and topoisomerase IV-DNA cleavage complexes in drug-treated cells. 3) To design novel drugs that overcome the most common quinolone resistance mutations in gyrase and topoisomerase IV. Studies will take great advan- tage of preliminary data and mechanistic (Aim 1), cellular (Aim 2), modeling, and competition studies. Our goal is to design drugs that do not rely on the water-metal ion bridge for their primary interaction with the bacterial type II enzymes and do not crossover into the human system. Three lead compounds have been identified. The primary research models for this study will be gyrase from Mycobacterium tuberculosis and gyrase and topoisomerase IV from E. coli and Staphylococcus aureus and to a lesser extent, Bacillus anthracis. Proposed research benefits greatly from previous studies from the Osheroff laboratory on the mechanism of bacterial and eukaryotic type II topoisomerases and the interaction of these enzymes with quinolones and other drugs.